Journal of Agricultural and Food Chemistry Q Copyright I972 by the American Chemical Soclaty
Volume 20, Number 6
NovemberlDecember 1972
Preparation and Characterization of Chlorinated Dibenzo-p-dioxins Albert E. Pohland” and George C. Y a n g
A series of 13 chlorinated dibenzo-p-dioxins containing from 1 to 8 chlorine atoms was prepared for use as standards in the development of analytical methodology for the chlorinated di benzo-p-dioxins and for use in toxicological studies. The synthetic pathways to these materials are reviewed and discussed from the standpoint of yield, convenience, and purity of product. The chemical and physical
n the fall of 1957, millions of chickens in the Eastern and Midwestern United States died of a disease (Friedman e[ al., 1959) characterized by excessive fluid in the pericardial sac. The names “chick edema factor” (CEF) and “hydropericardium toxic factor” (HPTF) were therefore applied to the component(s) in the feed responsible for this symptom. Liver and kideny damage was also observed (Schultz, 1968). Similar outbreaks of the disease occurred in 1960 and 1969; a nondistillable fatty material in the feed (possibly a soapstock) was thought to contain the toxic factor (Firestone, 1971). The acute toxicity of C E F led to research on the isolation and identification of the causative agent. A chlorinated hexahydrophenanthrene was initially suspected (Wootton and Courchene, 1964; Wootton et al., 1962) but X-ray diffraction showed one component to be 1,2,3,7,8,9-hexachlorodibenzo-p-dioxin (Cantrell et al., 1969). It was later realized that a whole series of chlorinated dibenzo-p-dioxins was present in a wide variety of products. To date the following products have been implicated (Firestone, 1971): food-grade oleic acids and emulsifiers prepared from them, vegetable oil soapstocks, distillation residues from commercial fatty acid manufacture, and chlorophenols and products prepared from them.
I
Division of Chemistry and Physics, Food and Drug Administration, U. S. Department of Health, Education, and Welfare, Washington, D. C. 20204.
properties of the various chlorinated dibenzo-pdioxins, including their stability, color reactions, infrared, ultraviolet, nuclear magnetic resonance, and phosphorescence spectra, were investigated. The wavelengths associated with the observed phosphorescence and the triplet state lifetime were dependent upon the number of chlorine atoms and their positions on the dibenzo-p-dioxin nucleus.
The presence of such highly chlorinated compounds in the food chain is of concern because these compounds are generally fat soluble and nonbiodegradable, tending to concentrate in the food chain, and many of them are acutely toxic and even carcinogenic (Bionetics Research Laboratories, 1967). 2,3,7,8-Tetrachlorodibenzo-p-dioxin, a common contaminant of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), is extremely toxic to a wide variety of animal species (Buu-Hoi et a/., 1971a; Farquharson et al., 1957; Higgenbotham et ul., 1968; Milnes, 1971 ; Sparschu et a/., 1971) and causes malformations of legs and beaks in the chick embryo (Flick ef a[., 1965). Its extreme potency as an acnegen is the basis for a sensitive test for the compound (Bauer et al., 1961; Dugois and Colomb, 1957; Jones and Krizek, 1962; Kimmig and Schulz, 1957a,b; Schulz, 1957). To provide pure standards of the various chlorinated derivatives for use in methods development and toxicology studies, we have synthesized a series of chlorinated dibenzo-p-dioxins. This paper presents methods of synthesis, their utility, and physical and chemical properties of the series. Dibenzo-p-dioxin is the name approved by Chemical Abstracts for the basic ring system. Three numbering systems, illustrated below, have been applied to the dibenzo-pdioxin nucleus: I, the Chemical Abstracts system (and the system used by the authors); 11, a symmetrical, essentially unambiguous, and easily understood system; and 111, a system frequently used in the older literature. Although 75 chlorinated dibenzo-p-dioxins are theoretically possible, only J. AGR. FOOD CHEM., VOL. 20, NO. 6, 1972 1093
POHLAND, YANG
the 2-chloro, 2,7-dichloro, 2,3,7,8-tetrachloro, and the octachlorodibenzo-p-dioxin were recorded in the literature at the time this work began. We have reported the synthesis of nine additional chlorinated dibenzo-p-dioxins.
I
I1
I11
EXPERIMENTAL SECTION
Melting points were determined on a Fisher-Johns apparatus and are accurate to within f1 '. Infrared spectral data were obtained with a Perkin-Elmer 621 infrared spectrophotometer and KBr disks. Ultraviolet spectral data were obtained with a Perkin-Elmer Model 202 ultraviolet-visible spectrophotometer. Phosphorescence spectra were obtained with a Perkin-Elmer Model MPF-2A recording spectrofluorometer with phosphorescence attachment. Nmr data were obtained in CDCls with a Varian HA-100 spectrometer. Mass spectral data were recorded with an Atlas CH-4B mass spectrometer. Gas chromatographic data were obtained with a Packard Model 7823 gas chromatograph using a hydrogen flame detector. For comparison purposes, all compounds were run on the same day at 172" with 80 mlimin Nz flow rate and a 6 ft OV-101 column. Dibenzo-p-dioxin. This compound was prepared according to the procedure described by Gilman and Dietrich (1957). The product, obtained in 10% yield, was recrystallized twice from isopropyl alcohol, mp 122-123' [lit. mp 119-122" (Cullinane and Davies, 1936; Cullinane et a/., 1934; Gilman and Dietrich, 1957; Tomita, 1945; Tomita et a/., 1954)]. A sample prepared by sublimation at 85-90' (1 mm) melted a t 122-123"; XnlaxJreoH 228 ( E 20,500), 290 nm ( E 3700); Xnlax'-oct 230 ( E 9760), 290 nm (E 3570); nmr AA'BB' pattern centered at 6 6.81 (35"); glc retention time 1.77 min. 1-Chlorodibenzo-p-dioxin. In a 250-ml, single-necked, round-bottomed flask was placed 10.01 g (0.10 mol) of catechol, 11.22 g (0.20 mol) of KOH, 16 (0.088 mol) of 1,2,3trichlorobenzene, and 100 ml of DMSO. The mixture was heated to reflux under a slow stream of N2 for 5 hr. On cooling, the dark brown mixture was poured, with stirring, over 1 kg of ice. Filtration through 24-cm fluted filter paper yielded a tan solid which was washed with distilled water until the filtrate was neutral and dried (12.36 g, mp 80-90°, 56.6%). This material was chromatographed over neutral alumina and eluted with benzene to yield a colorless product (mp 103104") which was recrystallized twice from MeOH; mp 104.5105.5"; XmaxJfeoH 232 ( e 23,9601, 291 nm ( E 3500); X,maxz-oct 236 ( E 10,500), 291 nm ( E 3060); nmr superposition of ABCD and ABC patterns centered at about 6 6.85 (35"); glc retention time 3.68 min. Anal. Calcd for Cl2H,O2Cl: C, 65.92; H, 3.23. Found: C, 65.72; H, 3.25. 2-Chlorodibenzo-p-dioxin. The dipotassium salt of catechol was prepared as a green-brown solid by dissolving 11.01 g (0.1 mol) in 50 ml of H 2 0 containing 11.22 g (0.2 mol) of KOH. Flash evaporation of the solution yielded the potassium salt, which was dissolved immediately in 200 ml of DMSO in a 500-rn1, single-necked flask. After the addition of 36.3 g (0.2 mol) of 1,2,4-trichlorobenzene, the mixture was heated at 175" under N) for 15 hr. On cooling, the solution was poured, with stirring, over 1 kg of crushed ice and filtered through 24-cm fluted paper. The grey solid which was recovered was washed well with distilled water and dried (15.25 1094 J. AGR. FOOD CHEM., VOL. 20, NO. 6, 1972
g, 70.5 %, mp 79-85 '). Three successive recrystallizations of this material from MeOH yielded a colorless solid: mp 8889" [lit. mp 87-90" (Gilman and Dietrich, 1957)l; XrnaxMeOH 231 ( E 23,300), 293 nm ( e 4020); X,nax'-oct 234 ( E 11,400), 295 nm ( E 3690); nmr superposition of AA'BB' pattern centered at 6 6.78 on an ABC pattern centered at about 6 6.81 (35"); glc retention time 3.56 min. Anal. Calcd for C I Z H ~ O ~ C IC, : 65.92; H, 3.23. Found: C, 65.88; H, 3.20. 2,7-Dichlorodibenzo-p-dioxin.The potassium salt of 2,4dichlorophenol was prepared by dissolution of the phenol (81.55 g, 0.5 mol) in 200 ml of MeOH containing 28.05 g (0.5 mol) of KOH. The resultant solution was allowed to stand overnight; flash evaporation of the mixture then yielded an oil which was taken up in 50 ml of benzene and decolorized with activated carbon. Addition of 100 ml of hexane precipitated the colorless potassium salt. The potassium salt of 2,4-dichlorophenol (20 g, 0.1 mol) was heated in the presence of 5 g of copper powder in a vacuum sublimator at 250" and 10 mm pressure for 24 hr. A small amount of product dioxin sublimed onto the cold finger. This material was dissolved in 50 ml of benzene, extracted twice with 10 mi of 10% KOH, washed with 10 mi of H 2 0 , dried over anhydrous Na?S04, and evaporated to yield a light yellow solid. Two recrystallizations from MeOH yielded colorless crystals; mp 209-210" (lit. mp 201-203" (Gilman and Dietrich, 1957; Julia and Baillarge, 1953), 203-205" (Tomita ef al., 1959), 207" (Denivelle et al., 1960; Ueo, 1941); Xn,,,nfeoH 235 (e24,900), 298 nm ( e 5060); X,,,,xi-oCt 236 ( E 11,800), 299 nm (E 4290); nmr ABC pattern centered at 6 6.82 (60"); glc retention time 7.15 min. 2,8-Dichlorodibenzo-p-dioxin.The potassium salt of 2,4,4'trichloro-2'-hydroxydiphenyl ether (Irgasan DP300, Geigy Industrial Chemicals) was prepared by dissolution of the phenol (28.95 g, 0.1 mol) in 200 ml of benzene. After addition of 5.61 g (0.1 mol) of KOH the mixture was refluxed overnight and the water formed was removed with a DeanStark trap. The clear solution which resulted was cooled to precipitate the pure white potassium salt. The potassium salt (25.9 g, 0.08 mol) was intimately mixed with 5 g of copper powder in a 250-ml single-necked, roundbottomed flask. The mixture was heated at 320" for 4 hr. On cooling, the brown residue was extracted with 250 ml of hot benzene, and the benzene solution was filtered, partially evaporated, and cooled to allow precipitation of 16.78 g (82.6%) of light yellow solid, mp 143-150'. Two recrystallizations from benzene yielded a colorless solid, nip 150.5151"; X,,,axnfeoH 234 ( e 27,300), 297 nm (e 5120); X I n a x i - O C t 237 ( E 11,700), 299 nm ( e 4680); nmr ABC pattern centered at about 6 6.80 (60"). Glc indicated a pure compound with a retention time of 7.16 min. However, X-ray ditfraction data indicated the presence of a second phase of unknown origin. A n d . Calcd for ClpHsOpClp: C, 56.95; H, 2.39. Found: C, 57.22; H , 2.52. 2,3-Dichlorodibenzo-p-dioxin. The dipotassium salt of catechol was prepared as described for 2-chlorodibenzo-pSuccessive recrystallization from dioxin (yield 67.5 MeOH and isooctane yielded a colorless solid, mp 163-164"; XnlaxUeoH 232 (E 27,000), 306 nm ( e 3600); XlflaXZ-oCt 238 ( E 11,500), 298 nm ( E 3420); nmr AA'BB' pattern centered at 6 6.96 and a singlet at 6 7.02 due to the protons in the 1 and 4 positions (35"); glc retention time 7.54 min. A n d . Calcd for CI2H6O?CI4:C, 56.95; H, 2.39. Found: C, 57.02; H, 2.41.
z).
CHLORINATED DIBENZO-PDIOXINS
1,2,4-Trichlorodibenzo-p-dioxin. In a 500-ml, three-necked flask fitted with a condenser, N2 inlet tube, and magnetic stirrer was placed 5.5 g (0.05 mol) of catechol, 13.82 g (0.1 mol) of K 2 C 0 3 ,and 100 ml of acetone. The mixture was refluxed 0.5 hr, after which 13.05 g (0.05 mol) of 2,3,5,6tetrachloronitrobenzene in 100 ml of acetone was added. This mixture was refluxed 2 h r ; 100 ml of H 2 0 was added and the mixture was cooled to ambient temperature. The solid grey precipitate was removed by filtration (9.55 g, 41.4%, mp 125-128"). Two recrystallizations of this material from acetone yielded a colorless product, mp 128-129"; XmaxlUeoH 239 ( E 24,800), 295 nm ( E 2200); 247 ( E 11,600), 290 nm ( E 2290); nmr broad singlet at 6 6.88 due to the four protons of the substituted ring and a sharp singlet at 8 7.00 due to the protons in the 3 position (60"); glc retention time 13.6 min. Anal. Calcd for ClsH502C1: C, 50.13; H, 1.75. Found: C, 50.24; H , 1.92. 2,3,7,8-Tetrachlorodibenzo-p-dioxin. This material was synthesized as described by Sanderman er al. (1957). Five recrystallizations from hot anisole yielded colorless needles, mp 305-306" [lit. mp 320-325" (Sanderman et a/., 1957), 298-300" (Milnes, 1971), 295" (Tomita er a/., 1959), 300-302" (Dietrich, 1957), 302--304" (Elvidge, 1971)l; nmr singlet a t 6 6.97 (66"); glc retention time 32.3 min. 1,3,6,8-Tetrachlorodibenzo-p-dioxin.The potassium salt of 2,4,6-trichlorophenoI was prepared by dissolving the phenol in water containing an equivalent amount of KOH, followed by filtration and evaporation of the filtrate to dryness on a flash evaporator. The potassium salt (10 g, 0.04 mol) was mixed intimately with 10 g of powdered copper and 10 g of K 2 C 0 3and the mixture was heated in a vacuum sublimation apparatus a t 300-320" at 10 mm of Hg for 5 hr. The white sublimate that had formed on the cold finger was dissolved in 50 ml of benzene; this solution was extracted with three 10-ml portions of 10% KOH, washed with 10 ml of HzO, and evaporated to yield a colorless solid (2.03 g, 15.3%). Recrystallization three times from benzene-methanol yielded long colorless needles, mp 219-219.5'; nmr AB pattern with V A a t 6 6.90 and i ' a ~t 6 7.02 and JAB = 2.4 Hz (66"); glc retention time 32.3 min. Anal. Calcd for Cl2H4O2CI4: C, 44.76; H , 1.25. Found: C, 45.11; H , 1.31. 1,2,3,4-Tetrachlorodibenzo-p-dioxin.This compound was prepared as described for 1,2,4-trichlorodibenzo-(j-dioxin; yield was 59.6 %. Two recrystallizations from acetone yielded long colorless needles, mp 189"C, that turned pink on exposure to fluorescent lights: nmr broad singlet centered a t 6 6.96 (60"); glc retention time 31.1 min. Ana/. Calcdfor CI2H4O2Cl4:C, 44.76; H , 1.25. Found: C, 44.45; H , 1.27. 1,2,3,4,7-Pentachlorodibenzo-p-dioxin. In a 250-rn1, singlenecked flask fitted with a magnetic stirrer was placed 1.63 g 1 g of (0.005 mol) of 1,2,3,4-tetrachlorodibenzo-p-dioxin, FeC13, 100 ml of CHC13, and a crystal of I?. A solution (20 ml) of C12 in CCll (0.5 M ) was added and the mixture was stirred at room temperature overnight; then it was filtered, washed twice with 20 ml of 2 0 z NaHSO, and 50 ml of H 2 0 , and flash evaporated to yield a colorless solid (1.67 g, 9 4 z , mp 180-1 90 "). Recrystallization from benzene-methanol yielded a colorless product, mp 195-196", >95% purity by glc; nmr ABC pattern centered at about 6 6.96 (65"). A n d . Calcd for C12H302C15:C, 40.44; H, 0.85. Found: C, 40.21 ; H, 0.88. 1,2,3,4,7,8-Hexachlorodibenzo-p-dioxin. In a 250-ml,
single-necked flask fitted with a magnetic stirrer was placed 1g 2.42 g (0.0075 mol) of 1,2,3,4-tetrachlorodibenzo-p-dioxin, of FeCI3, 100 ml of CHCI,, and a crystal of 12. A solution (45 ml) of Cln in cc14(0.5 M ) was added and the mixture was stirred a t room temperature for 5 days. The precipitate which formed was removed by filtration and dried (2.33 g, 87.6 mp 220-225"). Recrystallization of this material twice from chloroform and once from anisole yielded a colorless solid, mp 275", of >95 purity by glc. Anal. Calcd for C12H202Cle: C, 36.87; H, 0.52. Found: C, 36.55; H, 0.55. 1,2,4,6,7,9-HexachIorodibenzo-p-dioxin.This compound was prepared as described for 1,3,6,8-tetrachlorodibenzo-pdioxin except that the potassium salt was dried under vacuum (1 mm) for 24 hr after the first flash evaporation. Yield was 1.61 g (10.9 %) of 1,2,4,6,7,9-hexachlorodibenzo-p-dioxin, mp 200-210". Recrystallization from benzene-methanol three times yielded a colorless solid: mp 238-240"; glc retention time 95.4 min; nmr singlet at 6 7.18 (66"). The product was of 90% purity with a small amount of material whose retention time would indicate a pentachloro derivative. Anal. Calcd for C12H202Cle:C, 36.87; H, 0.52. Found: C, 37.11; H, 0.55. Octachlorodibenzo-p-dioxin. The general procedure described by Kulka (1961) was used to prepare this compound; yield 8 6 z ; mp 330" [lit. mp 326" (Denivelle et al., 1960), 318-319" (Tomita et al., 1959), 328-331" (Sanderman et al., 1957), 330-332" (Kulka, 1961), 324" (Denivelle et al., 1959)l.
z,
z
RESULTS
Many procedures for the synthesis of the parent ring system, dibenzo-p-dioxin, have been reported (Cullinane and Davies, 1936; Denivelle et al., 1960; Farbenfabriken, 1909; Gilman and Dietrich, 1957; Inubushi et a/., 1958; Julia and Baillarge, 1953; Keimatso and Yamaguchi, 1936; Tomitd, 1936, 1945; Tomita et a/., 1954). The most common procedure involves the condensation of 2 mol of the alkali metal salt of an o-halophenol at high temperature in the presence of a catalyst, usually copper powder or a copper alloy. This is a case of aromatic nucleophilic substitution, and yields are generally low (15-53 We have routinely used the method of Gilman and Dietrich (1957) to prepare dibenzo-p-dioxin itself; o-chlorophenol is heated in the presence of potassium carbonate and copper powder, and the black tarry product is refluxed with aqueous potassium hydroxide and extracted with ether. The resulting crude dioxin is purified by chromatography on alumina followed by vacuum sublimation. The utility of the method for general use with chlorinated phenols was limited by the formation of complex mixtures of products, presumably because the chlorophenols were not pure but were isomeric mixtures, and because chlorophenols oxidize easily (Musso, 1963). However, several modifications greatly increased the utility of the method. In one modification, use of vacuum sublimation to isolate the product facilitated separation of the chlorodioxin from the other products obtained. The potassium salt of the appropriate chlorophenol was heated at 350" with copper powder or potassium carbonate in a vacuum sublimation apparatus a t 10 mm pressure for 12 hr and the dioxin was sublimed onto the cold finger. The sublimate was dissolved in benzene and the solution was washed with 10% K O H and then water, dried (Na2S04),and finally flash evaporated. In every case the crude vacuum sublimate had to be successively recrystal-
z).
J. AGR. FOOD CHEM., VOL. 20, NO. 6 , 1972 1095
POHLAND, YANG
Table I. Synthesis of Chlorodioxins from Chlorophenols by Sublimation
Q f
3 c1
Chlorophenol 2,4-Dichloro2,4,6-Trichloro2,3,5,6-TetrachloroPentachloro-
aIn \
cu
Chlorodibenzo-pYield, dioxin 2,7-Dichloro10 1,3,6,8-Tetrachloro15.3 1,2,4,6,7,9-Hexachloro- 10.9 Octachloro1 5
z
Melting point, "C 209-210 219-220 238-240 330-331
Table 11. Synthesis of Chlorodioxins from Chlorobenzenes by Reaction with Catechol
Chlorobenzene 1.2,4-Trichloro1.2.3-Trichloro1,2,4.5-Tetrachloro-
Dioxin Yield, 2-Chloro70 5 1-Chloro56 6 2,3-Dichloro67 5
Melting point, "C 88-89 104 5-105 5 163-164
lized to attain a product of satisfactory purity. The results are shown in Table I. Although earlier workers had reported that pyrolysis of the silver or sodium salts of trichlorophenol led to formation of polymeric products (Hunter and Seyfried, 1921; Hunter et al., 1916; Tomita et a/., 1959), we prepared a 1,3,6,8-tetrachloro derivative that was not polymeric (based upon mass spectral and infrared data) and gave the typical dibenzo-p-dioxin nucleus color reaction (Tomita and Ueda, 1963; Tomita et a/., 1959). The potassium salt of a substituted diphenyl ether was used to prepare 2,8-dichlorodibenzo-p-dioxin,as follows.
/qoncl clam
c1
?+
c1 0
Table 111. Synthesis of Nitro-Substituted Dioxins
c1
K
In a second major modification of the Gilman-Dietrich method (1957) the dipotassium salt of catechol reacted with a n appropriately substituted chlorobenzene in DMSO (Chandler et al., 1971; Cullinane et al., 1934) (Table 11). No dioxin product could be isolated from tetrachlorocatechol monohydrate, however, even under a wide variety of experimental conditions. Certain nitro-substituted dioxins could be prepared easily by the following reaction scheme (Loudon and McCapra, 1959; Roberts et a/., 1935; Tomita, 1945).
c1
Picryl chloride undergoes nucleophilic substitution with exceptional ease, presumably because of the extremely strong -I and
1096 J. AGR. FOOD CHEM., VOL. 20, NO. 6, 1972
Yield, Nitrobenzene Pentachloro2,3,5,6-Tetrachloro-
Dioxin 1,2,3,4-Tetrachloro1,2,4-Trichloro-
z
59,6 41.4
Melting point, "C
189 128-129
-R effects of the three nitro substituents.
A steric effect was observed, since the reaction was successful only when nitro groups occupied both positions ortho to the chlorine substituent. Presumably the phenolate anion reacts first to form the corresponding diphenyl ether, followed by a second displacement reaction eliminating the nitro function (Turpin, 1891). This reaction was successfully applied to the condensation of catechol with various nitrohalobenzenes (Table III), but failed when 2,3-dichloronitrobenzene or tetrachlorocatechol was used. The highly toxic 2,3,7,8-tetrachlorodibenzo-p-dioxinwas prepared in good yield by direct chlorination of dibenzo-pdioxin in chloroform in the presence of ferric chloride and iodine (Sanderman et a/., 1957). This synthesis was successful presumably because the product precipitated from the reaction mixture due to low solubility in the chloroform solvent. However, by varying the reaction parameters it was possible to form the octachloro derivative by direct chlorination, but such reactions led to so many difficultly separable products that they were not useful synthetically. Theoretically one would expect attack by Cl+ to occur initially at the 2 position, considering the oxygen atom to be an o,p director and activator. Thus, the position para to the oxygen is activated toward electrophilic substitution by a strong resonance effect, as is the ortho position; however, the ortho position is deactivated by the I effect of the oxygen more than is the para position. Consequently direct chlorination should lead initially to the 2-chloro isomer. The position of attack of the second C1+ is an intriguing theoretical problem, however. It should enter at the 7 or 8 position, since the chlorinated ring is deactivated. The evidence in the literature, based largely upon the melting point of the isolated product, indicates that the 2,7-dichloro isomer is the major product (Gilman and Dietrich, 1957). The 2,8dichloro isomer may be more soluble and thus is not isolated. Although the two isomers have not been separated by glc, the structure of the 2,7-dichloro isomer is not in doubt, since the same material has been prepared by three independent synthetic pathways : direct chlorination of dibenzo-p-dioxin (Gilman and Dietrich, 1957), pyrolysis of the sodium salt of 2,4-dichlorophenol (Denivelle et ai.,1960; Julia and Baillarge, cia a Sandmeyer 1953), and from 2,7-diaminodibenzo-p-dioxin reaction (Ueo, 1941). The bromination of dibenzo-p-dioxin yielded 22 of the 2,8-dibromo isomer but only 7 x of the 2,7-dibromo isomer (Gilman and Dietrich, 1957). These results were explained in terms of structures IV and V. The oxonium ion in IV would receive less stabilization from the 2-bromo substituent than the oxonium ion in V.
x
11'
V
CHLORINATED DIBENZO-p-DIOXINS WAVELENGTH [MICRONS)
100
80
~
5
2 E60 Lu V
Z
2 40
z
Z
Q
E 20 -0 4000
3500
3000
2000
2500
1800 WAVEl$MQR CM 1400 1200 1000 800 600
400
Figure 1. Infrared spectrum of 2,3,7,8-tetrachIorodibenzo-p-dioxin.Aromatic CH stretch : 3122 (uw), 3080 (wm), 3028 (w). C=C skeletal in-plane stretch: 1569 (s), 1493 (s sh), 1473 (vs), 1464 (vs), 1457 (s sh), 1394 (m). =COC= asymmetric stretch: 1327 (s), 1212 (s). CCC trigonal aromatic ring in-plane deformation 1173 (s). Aromatic ring breathing 1115 (s). CH out-of-plane deformation 876 (s sh), 870 (vs). Aromatic CCI stretch 789 (m)
Direct chlorination of 1,2,3,4-tetrachlorodibenzo-p-dioxin in the presence of ferric chloride and iodine yielded higher chlorinated products. By carefully controlling the amount of chlorine added, both a pentachloro and a hexachloro isomer were isolated, each in >95 purity (by glcj, with the presumed structures shown below. No scrambling of the chlorine atoms of the 1,2,3,4-tetrachloro isomer occurred
c1
c1
c1
CI
I
c1
Cl
under the conditions used, as demonstrated by refluxing the tetrachloro compound in chloroform for 4 days in the presence of ferric chloride and iodine; no rearranged product could be detected by glc. Direct chlorination of pentachlorophenol in 1,2,4-trichlorooctachlorodibenzo-p-dioxin. A freebenzene yielded 83 radical mechanism has been proposed for this reaction, described by Kulka (1961). MOLECULAR STRUCTURE
The physical properties of these 13 chlorinated dibenzo-pdioxins were carefully investigated. The evidence for the structures of the individual isomers rests primarily upon the method of synthesis, correct elemental analysis, and reasonable infrared, ultraviolet, nuclear magnetic resonance, and mass spectra. In addition, all of the isomers gave the specific blue color reaction characteristic of the dibenzo-p-dioxin ring system when treated with concentrated sulfuric acid and a n oxidizing agent such as potassium nitrate (Tomita and Ueda, 1963; Tomita et al., 1959). The purity of each isomer was monitored during preparation by glc (flame ionization detector).
Table IV.
Ultraviolet Spectral Data for Chlorinated Dibenzo-p-dioxins CHC13 ~ _ _ _ ~ Dibenzo-p-dioxin x c x e’ e!€ ’ 293 3680 0.278 Unsubstituted 248 1020 248 1320 294 3190 1 -Chloro0.414 2-Chloro248 1140 299 3700 0.308 2,7-Dichloro247 1340 302 4590 0.292 2,8-Dichloro247 1180 299 4690 0.252 0.573 2,3-Dichloro247 1830 304 3190 2.36 253 5290 294 2250 1,2,4-Trichloro2.75 1,2,3,4-Tetrachloro257 6290 317 2290 310 5590 0.532 2,3,7&Tetrachloro248 2970 305 3440 1.61 1,3,6,8-Tetrachloro250 5540 2.20 259 5920 306 2690 1,2,3,4,7-Pentachloro1.47 1,2.3,4,7,8-Hexachloro- 259 5370 316 3660 310 1480 3.01 1,2,4,6,7,9-Hexachloro- 259 4450 5.48 Octachloro261 13150 318 2400 SPECTRAL PROPERTIES
The dibenzo-p-dioxin ring system was reported previously to exhibit a characteristically strong absorption band in the 1330-1280 cm-l region, presumably due to the asymmetric stretching of the COC bonds (Narisada, 1959). In this study the position of this absorption band depended upon the total number and position of chlorine substituents (1 327 cmin the 2,3,7,8-tetrachloro isomer, 1297 cm-I in the 1-chloro derivative, and 1002 cm-l in the octachloro derivative). Aromatic CC1 absorption was observed at 789 cm-’; other assignments are found in Figure 1. All of the isomers were distinguished easily from one another by their infrared spectra. The ultraviolet spectral data for the entire series of chlorinated dibenzo-p-dioxins are tabulated in Table IV. Dibenzop-dioxin absorbed maximally in chloroform at 248 nm ( e 1020) and 293 nm ( e 3680) (Wratten and Ali, 1967). A bathochromic shift of both absorption bands (Jaffe and Orchin, 1965a) was observed as more and more substituents were added to the dibenzo-p-dioxin nucleus. An unusual reversal of peak intensities was observed with chloroform as solvent but not with methanol or isooctane, where the short wavelength absorption band was always much more intense than the long wavelength band. J. AGR. FOOD CHEM., VOL. 20, NO. 6, 1972
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POHLAND, YANG
Table VI. Phosphorescence Decay Times of Chlorinated Dibenzo-p-dioxins Symmetrical molecules Time, sec Dibenzo-p-dioxin 0.54 2,7-Dichloro0.29 2,8-Dichloro0.22 1,3,6,8-TetrachIoro0.25 Octachloro0.25 Slightly unsymmetrical molecules 1-Chloro0.22 1,2,3,4,7-Pentachloro0.25 Unsymmetrical molecules 1,2,4-Trichloro0.14 1,2,3,4-Tetrachloro0.075 Table VII.
Mass Spectral Data for
Tetrachlorodibenzo-p-dioxins
+
410
450
x-
500
550
Figure 2. Phosphorescence spectra of 2,3,7,8- and 1,2,3,4-tetrachlorodibenzo-p-dioxins
Phosphorescence Spectral Data for Chlorodibenzo-p-dioxins Excitation, X Emission bands, Dibenzo-p-dioxin (nm) X (nm) Unsubstituted 294 386, 395,&410, 418, 426: 438 1-Chloro296 396, 402,a 422, 430a 2-Chloro295 400,a 420 2,7-Dichloro300 404,0 412, 426, 433, 458, 465 2,8-Dichloro304 400,a 404,b412, 420, 428, 432, 440, 450, 458 2,3-Dichloro312 408, 418, 435a 1,2,4-Trichloro304 412,b418: 427, 439a 1,2,3,4-Tetrachloro304 489 (very broad) 2,3,7,8-Tetrachloro305 411,a 418, 422, 436, 440, 448, 468 1,3,6,8-Tetrachloro300 410,a 418, 433, 438, 465 Octachloro310 433,a 458, 490 Table V.
Strongest peak.
Peak not resolved.
Intense phosphorescence was observed with dibenzo-pdioxin and all of the chlorinated derivatives. The excitation and emission maxima obtained in EPA (ethanol-isopentaneether, 2:5:5, Matheson, Coleman and Bell) a t 77°K are collected in Table V. Again a bathochromic shift in the absorption maxima was observed as the number of chlorine atoms present increased, presumably due to the admixture of halogen atomic orbitals with the aromatic orbitals, which resulted in increased separation of the singlet and triplet energy levels. The phosphorescence spectra of the 2,3,7,8-tetrachloro and of the 1,2,3,4-tetrachlorodibenzo-p-dioxinsare shown in Figure 2. In the symmetrical isomers, e.g., 2,3,7,8-tetrachloro, the emission peaks are relatively narrow and well resolved, indicating singlet-triplet transitions with resolved 1098 J. AGR. FOOD CHEM., VOL. 20, NO. 6, 1972
M M M M M 115 113 110 109 97 99 87 86 85 84 76 75 74 52 51
Ion mass (35~1)
("Cl) ( T I CO) 2(35C1) 2(35c1 c o )
+ +
50 a
1,3,6,Sa 100 15 45 5 40 12 10 10 40 45 20 18 5 36 20 2 10 71 2 2 45
Relative intensity, 2,3,7,8" 100 15 35 7 35 20 12 6 40 47 12 10 5 25 22 0 5
68 5 5 50
1,2,3,@ 100 7 40 10 50 5
15 0
I L7
10 15
2
2 4 23 23
25 33 25 55
Tetrachlorodibenzo-pdioxin (table prepared by J. N. Damico).
vibrational levels. Correlations with infrared and raman spectra are currently underway for identification of these phosphorescence substructures. With the highly unsymmetrical isomers, e.g., 1,2,3,4-tetrachloro, however, the emission band was broad and unresolved. In Table VI the phosphorescence decay times are tabulated. Due to the increase in spin-orbit coupling [the so-called heavy-atom effect (Jaffe and Orchin, 1965b)], the chlorinated dioxins have shorter triplet state lifetimes than does dibenzop-dioxin itself. A still further reduction in phosphorescence decay time is noted with the heavily unsymmetrical molecules. The chlorinated dibenzo-p-dioxins can be distinguished from the chlorinated dibenzofurans and chlorinated diphenyl ethers (Firestone et al., 1972) by their characteristic mass spectral fragmentation patterns (Firestone et al., 1972), in which the parent ion (M+) is most intense, but then apparently fragments, losing a chlorine atom (M+ - Cl), and CO [M (CO C1) and M - 2(CO Cl)]. In Table VI1 are compiled mass spectral data for three isomeric tetrachlorodibenzo-p-dioxins. The data show that the characteristic peaks arising from chlorine-containing fragments cannot distinguish between the isomers, since the relative intensities for these fragments are quite similar (BuuHoi et ai., 1971b). The 1,2,3,4-tetrachloro isomer, however,
+
+
CHLORINATED DIBENZO-p-DIOXINS
differs significantly from the other two isomers in relative intensities (underlined) in the low mass range, and it might be possible to distinguish it from the latter two under static conditions. The inability t o distinguish between isomeric chlorinated dibenzo-p-dioxins is similar t o that observed with tetrachlorobiphenyls (Safe and Hutzinger, 1971). Mass spectral data in conjunction with the nmr data differentiated among the three tetrachloro isomers. The nmr spectrum of the 1,3,6,8-tetrachIoro isomer exhibited an AB pattern with V A = 6 6.90 and V B = 6 7.02 and a coupling constant of 2.4 Hz characteristic for meta protons; the 2,3,7,8-tetrachloro isomer exhibited a single peak at 6 6.97. ACKNOWLEDGMENT
The authors are indebted to the following members of the FDA staff: Ernest Lustig, David Lincoln, and Elizabeth Hansen for preparation and interpretation of the nmr spectra; Joseph N. Damico for preparation and interpretation of the mass spectra; Jo-Yun T. Chen for infrared spectra; and Charles Graichen for elemental analysis. LITERATURE CITED
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